Host range mutants of vesicular stomatitis virus defective in in vitro RNA methylation.

Abstract

The viral RNA polymerase of detergent-treated vesicular stomatitis virus normally synthesizes viral mRNAs in vitro that are both guanylylated and methylated to give 5'-terminal 7mGpppAm caps. We have characterized a virus host range mutant, hr 1, that is totally defective in vitro in the methylation of mRNA, although full-length polyadenylylated mRNAs with 5' termini of the form GpppA are synthesized in normal yields. A second mutant, hr 8, is partially defective in methylation and synthesizes mRNAs in vitro with primarily GpppA and some GpppAm 5' termini. When used for in vitro translation, the unmethylated hr 1 mutant mRNA shows, as expected, reduced synthesis of viral proteins. These data provide direct evidence that the vesicular stomatitis virus-associated methyltransferase activities are virus encoded.

Identification of Sendai Virus L Protein Amino Acid Residues Affecting Viral mRNA Cap Methylation

“…Previous studies linked the inability of VSV cap methylation-defective mutants to grow in HEp-2 cells to a viral defect in mRNA cap guanine-N7 methylation and the consequent nontranslatability of primary VSV transcripts (16,17,24,25). It was also suggested that host cells methylate viral mRNA in permissive cell lines through an unknown mechanism (24).…”

“…Specifically, VSV mutants defective in cap methylation were temperature-sensitive (ts) and, more interestingly, host range restricted (hr), as manifested by their inability to grow in certain nonpermissive cell lines (e.g., HEp-2 cells) while retaining their ability to grow to high titers in permissive cells (e.g., BHK-21 cells) (16,17,24,25). Previous studies linked the inability of VSV cap methylation-defective mutants to grow in HEp-2 cells to the nontranslatability of primary VSV transcripts (24,25) and showed that host cells methylate viral mRNA in permissive cell lines through an unknown mechanism (24).…”

“…The methyltransferase (MTase) function of L was originally shown through the characterization of two host range (hr) mutants of vesicular stomatitis virus (VSV) (family Rhabdoviridae). It was shown that these mutants were defective in viral mRNA cap methylation (24,25) and that purified wild-type (wt) L protein was able to complement their defect during transcription in vitro, demonstrating that the VSV L protein possesses cap MTase activities (21). Later, independent computational analyses (5, 13) proposed that while L proteins share a very low degree of homology with the known S-adenosylmethionine (AdoMet)-dependent MTases at the amino acid level, their domain VI has a prototypical MTase fold, a glycine-rich motif shared by all members of the AdoMet-dependent MTase superfamily and directly involved in AdoMet binding (26,34), and several potential catalytic residues.…”

Sendai Virus RNA-dependent RNA Polymerase L Protein Catalyzes Cap Methylation of Virus-specific mRNA

“…In vesicular stomatitis virus (VSV), which possesses a negative strand RNA genome and belongs to the Rhabdoviridae family of the order Mononegavirales, cap formation has been suggested to proceed through the transfer of a GDP moiety from GTP to the 5Ј-monophosphate end of the mRNA to form the cap core structure from the in vitro transcription studies with purified virions (6). Finally, the cap core is methylated at the ribose-2Ј-OH position and then at the guanine-7N position to generate the cap I structure (7)(8)(9). However, direct biochemical studies on these reactions with purified viral component(s) have not been performed.…”

Cell-Type-Specific Growth Restriction of Vesicular Stomatitis Virus polR Mutants Is Linked to Defective Viral Polymerase Function

“…Such instances, however, can potentially identify critical host cell factors required for virus multiplication, and host range virus mutants are particularly useful in this regard. For example, VSV hr1 and hr8 mutants show defects in methylation of viral mRNA cap structures, a modification catalyzed by the virus L polymerase protein (18,22). The existence of such mutants implies that some cell types somehow compensate for the defective viral function, although which cellular activity is responsible is unknown (21).…”